Project Summary: The World Health Organization recently recommended the deployment of Wolbachia-infected mosquitoes for pilot biocontrol efforts that curb the transmission of Zika and dengue viruses to humans. This recommendation is based principally on two facets of Wolbachia biology. First, these maternally-transmitted, endosymbiotic bacteria cause pathogen blocking by altering the competency of mosquitoes to serve as effective hosts for viral replication. Second, Wolbachia alter sperm and egg via a process termed cytoplasmic incompatibility (CI) that is used for population suppression (reduction in mosquito population size) and population replacement (spread of pathogen-blocking Wolbachia into an uninfected population) strategies. Specifically, CI results in embryonic lethality when an infected male mates with an uninfected female. The modified sperm fertilize the uninfected egg, however post-fertilization defects ensue spanning failed replication of paternal DNA, delayed breakdown of paternal nuclear envelope and cell cycle activation, and a failure of segregation of paternal chromosomes. Importantly, the pre-fertilization modifications of sperm remain enigmatic, namely how Wolbachia in the testes express and use the toxin gene products of cytoplasmic incompatibility factors (cif) A and B to establish the onset of CI. As these two gene products represent a breakthrough in the understanding of CI, the central hypothesis of the proposed research is that prophage WO genes, cifA and cifB, encode protein products that escape the Wolbachia membrane in testes to interact with eukaryotic host ligands, ultimately modifying sperm integrity to cause CI. In Aim 1, I will use DNA cytochemistry and enzymatic assays to determine the types of sperm modifications inflicted by the Cif proteins in vivo. In Aim 2, I will investigate localization patterns and potential ligands to test the presumption that viral particles transport the proteins to interact with sperm nuclear DNA. Examinations thus far have yet to yield any mechanistic advance for the initial CI defects, and the rising interest in deploying Wolbachia to curb arbovirus transmission necessitates an explanation of Wolbachia's drive system. If successful, this research will pioneer mechanistic studies of Wolbachia-induced reproductive parasitism, inform Wolbachia's efficacy and delivery as a tool to control diverse zoonotic diseases, and provide multiple lines of evidence for the discovery of the biochemical basis of CI.